Novel multiplex stool-based assay for the detection of early-stage colon cancer in a Chinese population

Abstract

Background: Stool DNA (sDNA) methylation analysis is a promising, noninvasive approach for colorectal cancer screening; however, reliable biomarkers for detecting early-stage colon cancer (ECC) are lacking, particularly in the Chinese population.

Aim: To identify a novel stool-based assay that can improve the effectiveness of ECC screening.

Methods: A blinded case-control study was performed using archived stool samples from 125 ECC patients, and 125 control subjects with normal colonoscopy. The cohort was randomly divided into training and test sets at a 1.5:1 ratio. Targeted bisulfite sequencing (TBSeq) was conducted on five pairs of preoperative and postop-erative sDNA samples from ECC patients to identify DNA methylation biomarkers, which were validated using pyrosequencing. By logistic regression analysis, a multiplex stool-based assay was developed in the training set, and the detection performance was further assessed in the test set and combined set. The χ ² test was used to investigate the association of detection sensitivity with clinico-pathological features.

Results: Following TBSeq, three hypermethylated cytosine-guanine sites were selected as biomarkers, including paired box 8, Ras-association domain family 1 and secreted frizzled-related protein 2, which differed between the groups and were involved in important cancer pathways. An sDNA panel containing the three biomarkers was constructed with a logistic model. Receiver operating characteristic (ROC) analysis revealed that this panel was superior to the fecal immunochemical test (FIT) or serum carcinoembryonic antigen for the detection of ECC. We further found that the combination of the sDNA panel with FIT could improve the screening effectiveness. In the combined set, the sensitivity, specificity and area under the ROC curve for this multiplex assay were 80.0%, 93.6% and 0.918, respectively, and the performance remained excellent in the subgroup analysis by tumor stage. In addition, the detection sensitivity did not differ with tumor site, tumor stage, histological differentiation, age or sex, but was significantly higher in T4 than in T1-3 stage tumors (P = 0.041).

Conclusion: We identified a novel multiplex stool-based assay combining sDNA methylation biomarkers and FIT, which could detect ECC with high sensitivity and specificity throughout the colon, showing a promising application perspective.

Keywords: Colon cancer; DNA methylation; Early screening; Fecal immunochemical test; Stool biomarker.

Figure 1

Flow diagram of the study design. Candidate methylation biomarkers were selected by targeted bisulfite sequencing and then validated using pyrosequencing. At last, a diagnostic model was constructed and evaluated. CSG: Cancer sample group; HSG: Healthy sample group; DMRs: Differentially methylated regions; DMSs: Differentially methylated sites; ROC: Receiver operating characteristic; sDNA: Stool DNA; FIT: Fecal immunochemical test; PAX8: Paired box 8; RASSF1: Ras-association domain family 1; SFRP2: Secreted frizzled-related protein 2; CpG: Cytosine-guanine; ECC: Early-stage colon cancer.

Figure 2

DNA methylation analysis by targeted bisulfite sequencing. A: Principal component analysis of the methylation profiles between cancer sample group (CSG) and healthy sample group (HSG); B: Comparison of methylation density between CSG and HSG; C: Comparison of methylation level distribution between CSG and HSG. CSG: Cancer sample group; HSG: Healthy sample group; PC: Principal component.

Figure 3

Differentially methylated region analysis and biomarkers discovery. A: Volcano plot of differentially methylated regions (DMRs) in cancer sample group (CSG) vs healthy sample group (HSG); B: The distribution of the identified DMRs in the genome in relation to cytosine-guanine islands (CGIs); C: Circos plot of 2531 candidate DMRs on each of the 22 autosomes and the X chromosome; D: Kyoto Encyclopedia of Genes and Genomes pathway analysis of 2062 DMR-related genes; E: Gene Ontology analysis of 2062 DMR-related genes; F: Venn diagram of the overlapping target DMRs among different signatures. ^aP < 0.05. DMRs: Differentially methylated regions; CGI: Cytosine-guanine islands.

Figure 4

The evaluation of diagnostic model based on pyrosequencing. A: Comparison of methylation percentage of the three target biomarkers between the patients and controls in training set; B: Receiver operating characteristic (ROC) curves comparing fecal immunochemical test (FIT), stool DNA (sDNA) panel and sDNA panel + FIT for the detection of early-stage colon cancer (ECC) in training set; C: ROC curves comparing FIT, sDNA panel and sDNA panel + FIT for the detection of ECC in test set; D: ROC curves comparing FIT, sDNA panel and sDNA panel + FIT for the detection of ECC in combined set; E: ROC curves comparing FIT and sDNA panel + FIT for the detection of stage I ECC in combined set; F: ROC curves comparing FIT and sDNA panel + FIT for the detection of stage II ECC in combined set. ^aP < 0.05. sDNA: Stool DNA; FIT: Fecal immunochemical test; ROC: Receiver operating characteristic; PAX8: Paired box 8; RASSF1: Ras-association domain family 1; SFRP2: Secreted frizzled-related protein 2.

Figure 5

Impact of clinicopathologic covariates on screening. A: Sensitivities of fecal immunochemical test (FIT), serum carcinoembryonic antigen (CEA) and stool DNA (sDNA) panel + FIT for the detection of early-stage colon cancer (ECC), according to tumor-node-metastasis stage or T stage; B: Sensitivities of FIT, serum CEA and sDNA panel + FIT for the detection of ECC, according to tumor site or histological differentiation. sDNA: Stool DNA; FIT: Fecal immunochemical test; CEA: Carcinoembryonic antigen.